Methods for isolating a target analyte from a heterogenous sample

ABSTRACT

The invention generally relates to methods of using compositions that include sets of magnetic particles, members of each set being conjugated to an antibody specific for a pathogen, and magnets to isolate a pathogen from a body fluid sample.

RELATED APPLICATIONS

The present application is a division of U.S. nonprovisional patentapplication Ser. No. 13/952,130, filed on Jul. 26, 2013 and set to issueas U.S. Pat. No. 9,476,812, which is a continuation-in-part of U.S.nonprovisional patent application Ser. No. 13/091,510, filed Apr. 21,2011, now U.S. Pat. No. 8,841,104, which claims priority to and thebenefit of U.S. provisional patent application Ser. No. 61/326,588 filedApr. 21, 2010. In addition, the '130 application claims the benefit ofand priority to U.S. provisional patent application Ser. No. 61/739,616,filed Dec. 19, 2012. The content of each application is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The invention generally relates to methods of using compositions thatinclude sets of magnetic particles and magnets to isolate a pathogenfrom a body fluid sample.

BACKGROUND

Blood-borne pathogens are a significant healthcare problem. A delayed orimproper diagnosis of a bacterial or fungal infection can result insepsis, a serious, and often deadly, inflammatory response to theinfection. Sepsis is the 10^(th) leading cause of death in the UnitedStates. Early detection of bacterial infections in blood is the key topreventing the onset of sepsis. Traditional methods of detection andidentification of blood-borne infection include blood culture andantibiotic susceptibility assays. Those methods typically requireculturing cells, which can be expensive and can take as long as 72hours. Often, septic shock will occur before cell culture results can beobtained.

Alternative methods for detection of pathogens, particularly bacteriaand fungi, have been described by others. Those methods includemolecular detection methods, antigen detection methods, and metabolitedetection methods. Molecular detection methods, whether involving hybridcapture or polymerase chain reaction (PCR), require high concentrationsof purified DNA for detection. Both antigen detection and metabolitedetection methods also require a relatively large amount of pathogensand have high limit of detection (usually >10⁴ CFU/mL), thus requiringan enrichment step prior to detection. This incubation/enrichment periodis intended to allow for the growth of bacteria or fungi and an increasein cell numbers to more readily aid in identification. In many cases, aseries of two or three separate incubations is needed to isolate thetarget bacteria or fungus. However, such enrichment steps require asignificant amount of time (e.g., at least a few days to a week) and canpotentially compromise test sensitivity by killing some of the cellssought to be measured.

There is a need for methods for isolating target analytes, such asbacteria, from a sample, such as a blood sample, without an additionalenrichment step. There is also a need for methods of isolating targetanalytes that are fast and sensitive in order to provide data forpatient treatment decisions in a clinically relevant time frame.

SUMMARY

The present invention provides methods for isolating pathogens in abiological sample. The invention allows the rapid detection of pathogenat very low levels in the sample; thus enabling early and accuratedetection and identification of the pathogen. The invention is carriedout using sets of magnetic particles, members of each set beingconjugated to a binding element, such as an antibody, that is specificfor a pathogen. The invention allows detection of pathogen in aheterogeneous biological sample at levels, for example, from about 10CFU/ml to about 1 CFU/ml or lower.

Methods of the invention involve introducing magnetic particles to abiological sample (e.g., a tissue or body fluid sample). The sample isincubated to allow the particles to bind to pathogen in the sample, anda magnetic field is applied to capture pathogen/magnetic particlecomplexes on a surface. Optionally, the surface can be washed with awash solution that reduces particle aggregation, thereby isolatingpathogen/magnetic particle complexes. A particular advantage ofcompositions of the invention is for capture and isolation of bacteriaand fungi directly from blood samples at low concentrations that arepresent in many clinical samples (as low as 1 CFU/ml of bacteria orfungi in a body fluid). Preferably, the magnetic particles comprise acapture moiety (i.e., pathogen binding element) that has one or moremagnetic particles attached to it.

In certain aspects, methods of the invention involve obtaining aheterogeneous sample including a pathogen, exposing the sample to acocktail including a plurality of sets of magnetic particles, members ofeach set being conjugated to a capture moiety specific for a pathogen,and separating particle bound pathogen from other components in thesample. Methods of the invention may further involve characterizing thepathogen. Characterizing may include identifying the pathogen by anytechnique known in the art. Exemplary techniques include sequencingnucleic acid derived from the pathogen or amplifying the nucleic acid.

Any class of capture moiety capable of specifically binding to apathogen is suitable for use in methods and compositions of theinvention. Suitable classes of capture moieties include, e.g.,antibodies, lectins, bacteriophages, antimicrobial agents, andoligonucleotides. Classes of capture moieties may include one or moredifferent types of that class, e.g. an antibody class may include one ormore types of antibodies that are specific to different pathogens. Theantibodies conjugated to the particles may be either monoclonal orpolyclonal antibodies. Methods of the invention may be used to isolateone or more pathogens from heterogeneous sample. In particularembodiments, the heterogeneous sample is a blood sample.

According to certain aspects, compositions of the invention include aplurality of sets of magnetic particles, in which each set is conjugatedwith capture moieties having different specificities for differentpathogens. The capture moieties conjugated to the magnetic particles maybe of the same class or different class. For example, one set of capturemoieties may be antibodies specific to a first pathogen, and the otherset of capture moieties may be other antibodies specific to a secondpathogen. In another example, one set of capture moieties may beantibodies specific to a first pathogen, and the other set of capturemoieties may be lectins specific to a second pathogen.

In other aspects, compositions of the invention include at least onemagnetic particle that is conjugated to at least two sets of capturemoieties, in which each set of capture moieties is specific for adifferent pathogen. The at least two sets of capture moieties conjugatedto a magnetic particle may be of the same class or different classes.For example, a magnetic particle may have two or more antibodiesspecific to different pathogens. In another example, a magnetic particlemay be conjugated to an antibody specific to a pathogen and a lectinspecific to a different pathogen.

Compositions of the invention may be provided such that each set ofparticles conjugated to capture moieties is present at a concentrationdesigned for detection of a specific pathogen in the sample. In certainembodiments, all of the sets are provided at the same concentration.Alternatively, the sets are provided at different concentrations. Forexample, the concentration ratio of one set of capture moieties relativeto a second set of capture moieties is at a ratio of 50:50. In otherembodiments, the concentration ratio ranges from 1:99 to 99:1.

To facilitate detection of the different sets of pathogen/magneticparticle complexes the particles may be differently labeled. Anydetectable label may be used with compositions of the invention, such asfluorescent labels, radiolabels, enzymatic labels, and others. Inparticular embodiments, the detectable label is an optically-detectablelabel, such as a fluorescent label. Exemplary fluorescent labels includeCy3, Cy5, Atto, cyanine, rhodamine, fluorescien, coumarin, BODIPY,alexa, and conjugated multi-dyes.

Methods of the invention may be used to isolate one or more pathogensfrom a sample. Methods of the invention may be used to isolate only grampositive bacteria from a sample. Alternatively, methods of the inventionmay be used to isolate only gram negative bacteria from a sample. Incertain embodiments, methods of the invention are used to isolate bothgram positive and gram negative bacteria from a sample.

In still other embodiments, methods isolate specific pathogens, such asbacteria or fungi, from a sample. Exemplary fungal species that may becaptured by methods of the invention include species from the Candidagenus, Aspergillus genus, and Cryptococcus genus. In particularembodiments, the specific fungal species include C. albicans, C.glabarata, C. parapsilosis, C. tropicalis, C. krusei, Cryptococcusneoformans, Cryptococcus gattii. Exemplary bacteria that may be capturedand isolated by methods of the invention include bacteria of theEscherichia genus, Listeria genus, Clostridium genus, Enterobacteriaceaefamily, Mycobacterium genus, Shigella genus, Borrelia genus,Campylobacter genus, Bacillus genus, Salmonella genus, Enterococcusgenus, Streptococcus genus (such as Pneumococcus), Acinetobacter genus,Strenotrophomonas genus, Pseudomonas genus, Neisseria genus, andHaemophilus genus. The method may also be used to detect the mecA gene,which is a bacterial gene associated with antibiotic resistance.

Methods of the invention are not limited to isolating pathogens from abody fluid. Methods of the invention may be designed to isolate othertypes of target analytes, such as bacteria, fungi, protein, a cell, avirus, a nucleic acid, a receptor, a ligand, or any molecule known inthe art.

Compositions used in methods of the invention may use any type ofmagnetic particle. Magnetic particles generally fall into two broadcategories. The first category includes particles that are permanentlymagnetizable, or ferromagnetic; and the second category includesparticles that demonstrate bulk magnetic behavior only when subjected toa magnetic field. The latter are referred to as magnetically responsiveparticles. Materials displaying magnetically responsive behavior aresometimes described as superparamagnetic. However, materials exhibitingbulk ferromagnetic properties, e.g., magnetic iron oxide, may becharacterized as superparamagnetic when provided in crystals of about 30nm or less in diameter. Larger crystals of ferromagnetic materials, bycontrast, retain permanent magnet characteristics after exposure to amagnetic field and tend to aggregate thereafter due to strongparticle-particle interaction. In certain embodiments, the particles aresuperparamagnetic particles. In other embodiments, the magneticparticles include at least 70% superparamagnetic particles by weight. Incertain embodiments, the superparamagnetic particles are from about 100nm to about 250 nm in diameter. In certain embodiments, the magneticparticle is an iron-containing magnetic particle. In other embodiments,the magnetic particle includes iron oxide or iron platinum.

Another aspect of the invention provides methods for isolating pathogenfrom a heterogeneous sample, that involve labeling pathogen from abiological sample with a cocktail including a plurality of sets ofmagnetic particles, members of each set being conjugated to an antibodyspecific for a pathogen, exposing the sample to a magnetic field toisolate pathogen conjugated to the particles, and isolating particlebound pathogen from other components of the sample. Methods of theinvention may further involve eluting pathogen from the particles.Methods of the invention may further involve characterizing thepathogen. Characterizing may include identifying the pathogen by anytechnique known in the art. Exemplary techniques include sequencingnucleic acid derived from the pathogen or amplifying the nucleic acid.

DETAILED DESCRIPTION

The invention generally relates to methods of using compositions thatinclude sets of magnetic particles, members of each set being conjugatedto an antibody specific for a pathogen, and magnets to isolate apathogen from a body fluid sample. Certain fundamental technologies andprinciples are associated with binding magnetic materials to targetentities and subsequently separating by use of magnet fields andgradients. Such fundamental technologies and principles are known in theart and have been previously described, such as those described inJaneway (Immunobiology, 6^(th) edition, Garland Science Publishing), thecontent of which is incorporated by reference herein in its entirety.While some techniques are described exemplifying the isolation andcapture of bacteria, one skilled in the art would readily recognize thatthe techniques are generally applicable to all pathogens includingfungi.

Composition used in methods of the invention may use any type ofmagnetic particle. Production of magnetic particles and particles foruse with the invention are known in the art. See for example Giaever(U.S. Pat. No. 3,970,518), Senyi et al. (U.S. Pat. No. 4,230,685), Dodinet al. (U.S. Pat. No. 4,677,055), Whitehead et al. (U.S. Pat. No.4,695,393), Benjamin et al. (U.S. Pat. No. 5,695,946), Giaever (U.S.Pat. No. 4,018,886), Rembaum (U.S. Pat. No. 4,267,234), Molday (U.S.Pat. No. 4,452,773), Whitehead et al. (U.S. Pat. No. 4,554,088), Forrest(U.S. Pat. No. 4,659,678), Liberti et al. (U.S. Pat. No. 5,186,827), Ownet al. (U.S. Pat. No. 4,795,698), and Liberti et al. (WO 91/02811), thecontent of each of which is incorporated by reference herein in itsentirety.

Magnetic particles generally fall into two broad categories. The firstcategory includes particles that are permanently magnetizable, orferromagnetic; and the second category includes particles thatdemonstrate bulk magnetic behavior only when subjected to a magneticfield. The latter are referred to as magnetically responsive particles.Materials displaying magnetically responsive behavior are sometimesdescribed as superparamagnetic. However, materials exhibiting bulkferromagnetic properties, e.g., magnetic iron oxide, may becharacterized as superparamagnetic when provided in crystals of about 30nm or less in diameter. Larger crystals of ferromagnetic materials, bycontrast, retain permanent magnet characteristics after exposure to amagnetic field and tend to aggregate thereafter due to strongparticle-particle interaction. In certain embodiments, the particles aresuperparamagnetic particles. In certain embodiments, the magneticparticle is an iron containing magnetic particle. In other embodiments,the magnetic particle includes iron oxide or iron platinum.

In certain embodiments, the magnetic particles include at least about10% superparamagnetic particles by weight, at least about 20%superparamagnetic particles by weight, at least about 30%superparamagnetic particles by weight, at least about 40%superparamagnetic particles by weight, at least about 50%superparamagnetic particles by weight, at least about 60%superparamagnetic particles by weight, at least about 70%superparamagnetic particles by weight, at least about 80%superparamagnetic particles by weight, at least about 90%superparamagnetic particles by weight, at least about 95%superparamagnetic particles by weight, or at least about 99%superparamagnetic particles by weight. In a particular embodiment, themagnetic particles include at least about 70% superparamagneticparticles by weight.

In certain embodiments, the superparamagnetic particles are less than100 nm in diameter. In other embodiments, the superparamagneticparticles are about 150 nm in diameter, are about 200 nm in diameter,are about 250 nm in diameter, are about 300 nm in diameter, are about350 nm in diameter, are about 400 nm in diameter, are about 500 nm indiameter, or are about 1000 nm in diameter. In a particular embodiment,the superparamagnetic particles are from about 100 nm to about 250 nm indiameter.

In certain embodiments, the particles are particles (e.g.,nanoparticles) that incorporate magnetic materials, or magneticmaterials that have been functionalized, or other configurations as areknown in the art. In certain embodiments, nanoparticles may be used thatinclude a polymer material that incorporates magnetic material(s), suchas nanometal material(s). When those nanometal material(s) orcrystal(s), such as Fe₃O₄, are superparamagnetic, they may provideadvantageous properties, such as being capable of being magnetized by anexternal magnetic field, and demagnetized when the external magneticfield has been removed. This may be advantageous for facilitating sampletransport into and away from an area where the sample is being processedwithout undue particle aggregation.

One or more or many different nanometal(s) may be employed, such asFe₃O₄, FePt, or Fe, in a core-shell configuration to provide stability,and/or various others as may be known in the art. In many applications,it may be advantageous to have a nanometal having as high a saturatedmoment per volume as possible, as this may maximize gradient relatedforces, and/or may enhance a signal associated with the presence of theparticles. It may also be advantageous to have the volumetric loading ina particle be as high as possible, for the same or similar reason(s). Inorder to maximize the moment provided by a magnetizable nanometal, acertain saturation field may be provided. For example, for Fe₃O₄superparamagnetic particles, this field may be on the order of about 0.3T.

The size of the nanometal containing particle may be optimized for aparticular application, for example, maximizing moment loaded upon atarget, maximizing the number of particles on a target with anacceptable detectability, maximizing desired force-induced motion,and/or maximizing the difference in attached moment between the labeledtarget and non-specifically bound targets or particle aggregates orindividual particles. While maximizing is referenced by example above,other optimizations or alterations are contemplated, such as minimizingor otherwise desirably affecting conditions.

In an exemplary embodiment, a polymer particle containing 80 wt % Fe₃O₄superparamagnetic particles, or for example, 90 wt % or highersuperparamagnetic particles, is produced by encapsulatingsuperparamagnetic particles with a polymer coating to produce a particlehaving a diameter of about 250 nm.

Compositions of the invention include sets of a plurality of magneticparticles specific to different targets of interest (e.g., pathogens).Each set of magnetic particles has a target-specific binding moiety(capture moiety) that allows for each set to specifically bind thetarget of interest in the heterogeneous sample. Exemplary classes ofcapture moieties include oligonucleotides (including nucleic acidprobes), proteins, ligands, lectins, antibodies, aptamers,bactertiophages, host innate immunity biomarkers (e.g., CD14), hostdefense peptides (e.g., defensins), bacteriocins (e.g., pyocins), andreceptors. The capture moiety may be specific to a certain specieswithin a genus of pathogen, or the capture moiety may be generallyspecific to several species within or the entire genus of pathogen. Aclass of capture moieties may include one or more different types ofthat class, e.g. an antibody specific to one pathogen and an antibodyspecific to another pathogen. In addition, one set of magnetic particlesmay be conjugated to a class of capture moieties that are different froma class of capture moieties conjugated to another set. For example, oneset of magnetic particles may be conjugated to an antibody specific to apathogen and another set of magnetic particles may be conjugated to alectin specific to a different pathogen. The classes and types ofcapture moieties utilized will depend on the target to be captured andisolated.

In certain embodiments, each magnetic particle of a set is conjugated toat least two different sets of capture moieties specific to differentpathogens. That is, one magnetic particle may have two or more capturemoieties specific to different pathogens. The two or more capturemoieties conjugated to a magnetic particle may be of the same class ordifferent class. For example, a magnetic particle may have two or moreantibodies specific to different pathogens. In another example, amagnetic particle may be conjugated to an antibody specific to apathogen and a lectin specific to a different pathogen.

In particular embodiments, the capture moiety is an antibody, such as anantibody that binds a particular pathogen. General methodologies forantibody production, including criteria to be considered when choosingan animal for the production of antisera, are described in Harlow et al.(Antibodies, Cold Spring Harbor Laboratory, pp. 93-117, 1988). Forexample, an animal of suitable size such as goats, dogs, sheep, mice, orcamels are immunized by administration of an amount of immunogen, suchthe target bacteria, effective to produce an immune response. Anexemplary protocol is as follows. The animal is injected with 100milligrams of antigen resuspended in adjuvant, for example Freund'scomplete adjuvant, dependent on the size of the animal, followed threeweeks later with a subcutaneous injection of 100 micrograms to 100milligrams of immunogen with adjuvant dependent on the size of theanimal, for example Freund's incomplete adjuvant. Additionalsubcutaneous or intraperitoneal injections every two weeks withadjuvant, for example Freund's incomplete adjuvant, are administereduntil a suitable titer of antibody in the animal's blood is achieved.Exemplary titers include a titer of at least about 1:5000 or a titer of1:100,000 or more, i.e., the dilution having a detectable activity. Theantibodies are purified, for example, by affinity purification oncolumns containing protein G resin or target-specific affinity resin.

Polyclonal antibodies, monoclonal antibodies, or both can be conjugatedto magnetic particles in accordance with compositions and methods of theinvention. Polyclonal antibodies are antibodies that are secreted bydifferent B cell lineages, and are a collection of immunoglobulinmolecules that react against a specific antigen, each identifying adifferent epitope. Thus, polyclonal antibodies recognize multipleepitopes on any one antigen. Polyclonal antibodies are useful inidentifying homologous pathogens, and allow for general isolation andcapture of a range of species. In contrast, monoclonal antibodies areconstructed from one cell line, and recognize only one epitope on anantigen. Monoclonal antibodies are more specific, and typically onlybind to the epitope of the specific target cell (e.g. less likely tobind to a range of species).

The technique of in vitro immunization of human lymphocytes is used togenerate monoclonal antibodies. Techniques for in vitro immunization ofhuman lymphocytes are well known to those skilled in the art. See, e.g.,Inai, et al., Histochemistry, 99(5):335 362, May 1993; Mulder, et al.,Hum. Immunol., 36(3):186 192, 1993; Harada, et al., J. Oral Pathol.Med., 22(4):145 152, 1993; Stauber, et al., J. Immunol. Methods,161(2):157 168, 1993; and Venkateswaran, et al., Hybridoma, 11(6) 729739, 1992. These techniques can be used to produce antigen-reactivemonoclonal antibodies, including antigen-specific IgG, and IgMmonoclonal antibodies.

Any antibody or fragment thereof having affinity and specific for thebacteria of interest is within the scope of the invention providedherein. Immunomagnetic particles against Salmonella are provided inVermunt et al. (J. Appl. Bact. 72:112, 1992). Immunomagnetic particlesagainst Staphylococcus aureus are provided in Johne et al. (J. Clin.Microbiol. 27:1631, 1989). Immunomagnetic particles against Listeria areprovided in Skjerve et al. (Appl. Env. Microbiol. 56:3478, 1990).Immunomagnetic particles against Escherichia coli are provided in Lundet al. (J. Clin. Microbiol. 29:2259, 1991).

In certain embodiments, the target-specific binding moiety is a lectin.Lectins are sugar-binding proteins that are highly specific for theirsugar moieties. Exemplary lectins that may be used as target-specificbinding moieties include Concanavalin (ConA), Wheat Germ Extract WGA).Lectins that specifically bind to bacteria and fungi are known, see,e.g. Stoddart, R. W., and B. M. Herbertson. “The use offluorescein-labelled lectins in the detection and identification offungi pathogenic for man: a preliminary study.” Journal of medicalmicrobiology 11.3 (1978): 315-324; and U.S. Pat. No. 5,004,699. Inaddition, other lectins that have pathogen-binding properties are shownin Table 1 below.

TABLE 1 Lectins with Carbohydrate Specificity Lectin Source CarbohydrateSpecificity AMA Arum maculatum Mannose (AMA) from ‘lords and ladies’flower ASA Allium sativum Mannose (ASA) from garlic Con-A Canavaliaensiformis α-D-Mannose, (Con-A) from jack α-D-Glucose, branched beanmannose GS-II Griffonia simplicoflia Terminal α- and β- (GS-II) fromshrub GS N-Acetylglucosamine HHA Hippeastrum hybrid Mannonse (int and(HHA) from amaryllis term residues) IRA Iris hybric (IRA)N-Acetyl-D-Galactosamine from Dutch Iris LEA Lycopersicon esculentumβ(1,4)-linked (LEA) from tomato N-Acetylglucosamine LPA Limuluspolyphemus Sialic Acid (LPA) from horseshoe crab (N-Acetylneuraminicacid) MIA Mangidera indica Exact specificity unknown (MIA) from mangoPAA Perseau americana Exact specificity unknown (PAA) from avocado WGATriticum vulgaris (WGA) (GlcNAc-β-(1,4)-GlcNAc)1-4>β- from Wheat GermGlcNAc>Neu5Ac WGA-S Succinyl Triticum vulgare(GlcNAc-β-(1,4)-GlcNAc)1-4>β- (WGA-S) from wheat germ GlcNAc>Neu5Ac

Capture moieties suitable for use in methods of the invention may alsoinclude a nucleic acid ligand (aptamer). A nucleic acid ligand (aptamer)is a nucleic acid macromolecule (e.g., DNA or RNA) that binds tightly toa specific molecular target Like all nucleic acids, a particular nucleicacid ligand may be described by a linear sequence of nucleotides (A, U,T, C and G), typically 15-40 nucleotides long. In solution, the chain ofnucleotides forms intramolecular interactions that fold the moleculeinto a complex three-dimensional shape. The shape of the nucleic acidligand allows it to bind tightly against the surface of its targetmolecule. In addition to exhibiting remarkable specificity, nucleic acidligands generally bind their targets with very high affinity, e.g., themajority of anti-protein nucleic acid ligands have equilibriumdissociation constants in the picomolar to low nanomolar range.

Nucleic acid ligands are generally discovered using an in vitroselection process referred to as SELEX (Systematic Evolution of Ligandsby EXponential enrichment). See for example Gold et al. (U.S. Pat. No.5,270,163). SELEX is an iterative process used to identify a nucleicacid ligand to a chosen molecular target from a large pool of nucleicacids. The process relies on standard molecular biological techniques,using multiple rounds of selection, partitioning, and amplification ofnucleic acid ligands to resolve the nucleic acid ligands with thehighest affinity for a target molecule.

In addition, the capture moiety may be a nucleic acid probe, typicallyan oligonucleotide, that specifically binds to a target nucleic acidsequence. A probe is generally a single-stranded nucleic acid sequencecomplementary to some degree to a nucleic acid sequence sought to bedetected (“target sequence”). A probe may be labeled with a reportergroup moiety such as a radioisotope, a fluorescent or chemiluminescentmoiety, or with an enzyme or other ligand which can be used fordetection. Standard techniques are used for nucleic acid and peptidesynthesis. These molecular biological techniques may be performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.,). Synthesis of complementary DNA is describedin, for example, Nature Methods 2, 151-152 (2005)doi:10.1038/nmeth0205-151. Background descriptions of the use of nucleicacid hybridization to detect particular nucleic acid sequences are givenin Kohne, U.S. Pat. No. 4,851,330 issued Jul. 25, 1989, and by Hogan etal., International Patent Application No. PCT/US87/03009, entitled“Nucleic Acid Probes for Detection and/or Quantitation of Non-ViralOrganisms,” both references hereby incorporated by reference herein.Hogan et al., supra, describe methods for determining the presence of anon-viral organism or a group of non-viral organisms in a sample (e.g.,sputum, urine, blood and tissue sections, food, soil and water).

Reporter phages may also be used as a capture moiety. Reporter phages,such as bacteriophages, are typically genetically modified phages usedto introduce a gene of interest into a host pathogen. The reporter geneincorporates detectable codes for a fluorescent or substrate dependentcolorimetric marker into host pathogen, which allows for subsequentpathogen detection. Bacteriophages used for pathogen detection aredescribed in detail in Singh, Amit, et al. “Bacteriophage based probesfor pathogen detection.” Analyst 137.15 (2012): 3405-3421.; Dover, JasonE., et al. “Recent advances in peptide probe-based biosensors fordetection of infectious agents.” Journal of microbiological methods 78.1(2009): 10-19.

Methods for attaching the target-specific binding moiety to the magneticparticle are known in the art. Coating magnetic particles withantibodies is well known in the art, see for example Harlow et al.(Antibodies, Cold Spring Harbor Laboratory, 1988), Hunter et al.(Immunoassays for Clinical Chemistry, pp. 147-162, eds., ChurchillLivingston, Edinborough, 1983), and Stanley (Essentials in Immunologyand Serology, Delmar, pp. 152-153, 2002). Such methodology can easily bemodified by one of skill in the art to bind other types oftarget-specific binding moieties to the magnetic particles. Certaintypes of magnetic particles coated with a functional moiety arecommercially available from Sigma-Aldrich (St. Louis, Mo.).

Since each set of particles is conjugated with capture moieties havingdifferent specificities for different pathogens, compositions used inmethods of the invention may be provided such that each set of capturemoiety conjugated particles is present at a concentration designed fordetection of a specific pathogen in the sample. In certain embodiments,all of the sets are provided at the same concentration. Alternatively,the sets are provided at different concentrations. For example,compositions may be designed such that sets that bind gram positivebacteria are added to the sample at a concentration of 2×10⁹ particlesper/ml, while sets that bind gram negative bacteria are added to thesample at a concentration of 4×10⁹ particles per/ml. Compositions usedwith methods of the invention are not affected by antibodycross-reactivity. However, in certain embodiments, sets are specificallydesigned such that there is no cross-reactivity between differentantibodies and different sets.

Methods of the invention may be used to isolate only gram positivebacteria from a sample. Alternatively, methods of the invention may beused to isolate only gram negative bacteria from a sample. In certainembodiments, methods of the invention are used to isolate both grampositive and gram negative bacteria from a sample. Such compositionsallow for isolation of essentially all bacteria from a sample.

In still other embodiments, compositions used with methods of theinvention are designed to isolate specific pathogen from a sample.Typically, the pathogens are fungi (e.g, yeast or other pathogenicfungi) or bacteria. Exemplary fungal species that may be captured bymethods of the invention include species from the Candida genus,Aspergillus genus, and Cryptococcus genus. In particular embodiments,the specific fungal species include C. albicans, C. glabarata, C.parapsilosis, C. tropicalis, C. krusei, Cryptococcus neoformans,Cryptococcus gattii. Exemplary bacteria that may be captured andisolated by methods of the invention include bacteria of the Escherichiagenus, Listeria genus, Clostridium genus, Enterobacteriaceae family,Mycobacterium genus, Shigella genus, Borrelia genus, Campylobactergenus, Bacillus genus, Salmonella genus, Enterococcus genus,Streptococcus genus (such as Pneumococcus), Acinetobacter genus,Strenotrophomonas genus, Pseudomonas genus, Neisseria genus, andHaemophilus genus. The method may also be used to detect the mecA gene,which is a bacterial gene associated with antibiotic resistance. Inaddition, the specific species of pathogen selected for capture andisolation may be based on a certain phenotypic characteristic. Forexample, capture moieties conjugated to a magnetic particle may bedesigned to capture coagulase-negative Staphylococcus species (e.g.species of Staphyloccous that inhibit blood clot formation).

The sets of magnetic particles may be mixed together to isolate certainfungi, bacteria, or both. These sets can be mixed together to isolatefor example, E. coli and Listeria; or E. coli, Listeria, andClostridium; or Mycobacterium, Campylobacter, Bacillus, Salmonella, andStaphylococcus, etc. One set may be specific to a certain bacterium andanother set may be specific to a certain fungus. Any combination of setsmay be used and compositions of the invention will vary depending on thesuspected pathogen or pathogens to be isolated. In certain embodiments,compositions include two, three, four, five . . . 10, 20, etc. differentsets of magnetic particles conjugated to different pathogens.

In preferred embodiments, sets of magnetic particles conjugated tocapture moieties that are specific to different targets withinmulti-plex detection panel. For example, sets of modified magneticparticles may be chosen to isolate two or more certain pathogens. Thetwo or more certain pathogens chosen may be causal of similar symptoms(e.g. digestive abnormalities), commonly found in the type of body fluid(e.g. stool), or pathogens common to a certain area (e.g. hospitalsetting). The following tables show exemplary panels of fungal (Table 2)targets and bacterial (Table 3) targets. “Sp” means a target specieswithin a genus, and “Spp” means multiple target species of genus.

TABLE 2 Fungal Panels—Compositions of the invention wil include capturemoieties bound to magnetic particles specific to each of the targets inthe panel. Fungi Assay Target 1 Target 2 Target 3 Target 4 Target 5Panel A Species of C. albicans C. glabrata C. parapsilosis N/A Candida(or C. genus tropicalis) Panel B Species of C. albicans C. glabrata C.parapsilosis/ C. krusei Candida C. tropicalis genus Panel C Species ofC. albicans C. glabrata/ C. parapsilosis/ Cryptococcus Candida C. kruseiC. tropicalis spp. genus Panel D Candida C. albicans/C. C. glabrata/C.Aspergillus spp. Cryptococcus genus parapsilosis/ krusei spp. C.tropicalis Panel E Cryptococcus C. albicans/C. C. glabrata/C.Aspergillus spp. Cryptococcus neoformans parapsilosis/C. krusei gattiitropicalis

TABLE 3 Bacteria Panels—Compositions of the invention will includecapture moieties bound to magnetic particles specific to each of thetargets in the panel. Bacteria Assay Target 1 Target 2 Target 3 Target 4Target 5 Panel S. aureus CoNS mecA Streptococcus N/A A-sp./Enterococcocus Gram sp. Positive Panel Panel EnterobacteriaceaePseudomonas Acinetobacter Stenotrophomonas N/A B- aeruginosa sp.maltophilia Gram (or other GNR Negative such as Panel Neisseria,Haemophilus) Panel Staphylococcus CoNS Enterobacteriaceae PseudomonasN/A C sp. aeruginosa Panel Staphylococcus Enterococcus E. coliPseudomonas N/A D sp. sp./Streptococcus aeruginosa sp. Panel EStaphylococcus CoNS Enterococcus Enterobacteriaceae N/A aureussp./Streptococcus sp. Panel F Staphylococcus EnterococcusEnterobacteriaceae Candida spp. N/A sp. sp./Streptococcus sp. Panel S.aureus CoNS mecA Streptococcus Enterococcocus G- sp. sp. Gram PositivePanel Panel Enterobacteriaceae Pseudomonas AcinetobacterStenotrophomonas Neisseria H- aeruginosa sp. maltophilia meningitidisGram Negative Panel Panel I Staphylococcus Enterococcus StreptococcusEnterobacteriaceae Pseudomonas sp. sp. sp. aeruginosa Panel JStaphylococcus Enterococcus Streptococcus E. coli Pseudomonas sp. sp.sp. aeruginosa Panel Staphylococcus CoNS Enterococcus EnterobacteriaceaePseudomonas K aureus sp./Streptococcus aeruginosa sp. Panel LStaphylococcus Enterococcus Enterobacteriaceae Pseudomonas Acinetobactersp. sp./Streptococcus aeruginosa sp. sp.

In certain embodiments, compositions of the invention include at leastone set magnetic particles conjugated to a capture moiety specific to aninternal control (IC). For example, an IC detectable marker may beplaced into a clinical sample suspected of containing a pathogen. Fordetection of the pathogen, a plurality of sets of magnetic particles areintroduced to that clinical sample, in which at least one set isconjugated to a capture moiety designed to bind the IC detectable markerand one or more other sets are conjugated to capture moieties designedto bind to the suspected pathogens. When the assay is conducted tocapture and isolate the pathogen, the presence or absence of the ICdetectable marker indicates whether the assay properly separated themarker from the sample. That is, the introduction of a detectable markerallows one to determine whether the sets of magnetic particlesconjugated to capture moieties are properly capturing or isolating thetarget pathogens that are present within the fluid. This is important todistinguish between a failed assay (i.e. failure to identify a targetpathogen present in the sample) and a positive assay (i.e. positivelydetermining the absence of a target pathogen). The presence of themarker indicates that the assay worked properly; and thus any detectionof pathogen (or the absence of the pathogen) is accurate and not theresult of a failed assay. Use of IC detectable markers is described inmore detail in co-owned and co-assigned U.S. Provisional Application No.61/739,577, filed Dec. 18, 2012. The panels listed in Tables 2 and 3above may include the addition of an IC detectable marker inserted intothe sample, and the sets of magnetic particles would include setsconjugated to capture moieties specific to targets listed in the panelas well as the internal control.

Methods of the invention that utilize the internal control involveobtaining a sample suspected of containing a pathogen and introducing adetectable maker into the sample. An assay is conducted to detect thesuspected pathogen and the detectable marker in the sample using aplurality of sets of magnetic particles (as described herein). Membersof at least one set of magnetic particles are conjugated to bindingentity specific to a pathogen, and members of at least one set ofmagnetic particles are conjugated to a binding entity specific to thedetectable marker. After the conducting step, the presence of absence ofthe marker in the sample is determined. Based on the presence or absenceof the detectable marker, a determination is made about the presence orabsence of targets in the sample. The presence of the marker indicatesthat the assay worked properly; and thus any detection of pathogen (orthe absence of pathogen) is accurate and not the result of a failedassay.

Any detectable marker may be used for an internal control. In certainembodiments, the IC detectable marker may be a microbe, including aviable or nonviable microbe. In addition, the IC detectable marker canbe sufficiently similar to the target such that the assay performsfunctionally in the same manner for the detectable marker and thetarget. The detectable marker may be labeled or otherwise modified toallow for their differentiation from targets originally present in thesample. For example, but not by way of limitation, the detectable markercan be genetically modified so as to express a fluorescent protein, oralternatively, the detectable marker could be pre-stained with apersistent stain to allow their differentiation from microbes that areoriginally present in the fluid composition. In addition, the detectablemarker may be chosen based a chromogen dye specific reaction to thepresence of the detectable marker.

Capture of a wide range of target microorganisms simultaneously can beachieved by utilizing antibodies specific to target class, such aspan-Gram-positive antibodies, pan-Gran-negative antibodies or antibodiesspecific to a subset of organisms of a certain class. Further, expandedreactivity can be achieved by mixing particles of different reactivity.It was shown in our experiments that addition of high concentration ofnon-specific particles does not interfere with the capture efficiency oftarget-specific particles. Similarly, several different particlepreparations can be combined to allow for the efficient capture ofdesired pathogens. In certain embodiments the particles can be utilizedat a concentration between 1×10⁸ and 5×10¹⁰ particles/mL.

In certain embodiments the expanded coverage can be provided by mixingantibodies with different specificity before attaching them to magneticparticles. Purified antibodies can be mixed and conjugated to activatedmagnetic particle using standard methods known in the art.

To facilitate detection of the different sets of pathogen/magneticparticle complexes the particles may be differently labeled. Anydetectable label may be used with compositions of the invention, such asfluorescent labels, radiolabels, enzymatic labels, and others. Thedetectable label may be directly or indirectly detectable. In certainembodiments, the exact label may be selected based, at least in part, onthe particular type of detection method used. Exemplary detectionmethods include radioactive detection, optical absorbance detection,e.g., UV-visible absorbance detection, optical emission detection, e.g.,fluorescence; phosphorescence or chemiluminescence; Raman scattering.Preferred labels include optically-detectable labels, such asfluorescent labels. Examples of fluorescent labels include, but are notlimited to, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid;acridine and derivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives; eosin, eosin isothiocyanate, erythrosin and derivatives;erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives; 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Atto dyes, Cy3; Cy5; Cy5.5; Cy7; IRD 700;IRD 800; La Jolta Blue; phthalo cyanine; and naphthalo cyanine.Preferred fluorescent labels are cyanine-3 and cyanine-5. Labels otherthan fluorescent labels are contemplated by the invention, includingother optically-detectable labels. Methods of linking fluorescent labelsto magnetic particles or antibodies are known in the art.

Methods of the invention may be used to isolate pathogen from anyheterogeneous sample. In particular embodiments, methods of theinvention isolate a pathogen from body fluid. A body fluid refers to aliquid material derived from, for example, a human or other mammal. Suchbody fluids include, but are not limited to, mucus, blood, plasma,serum, serum derivatives, bile, phlegm, saliva, sweat, amniotic fluid,mammary fluid, urine, sputum, and cerebrospinal fluid (CSF), such aslumbar or ventricular CSF. A body fluid may also be a fine needleaspirate. A body fluid also may be media containing cells or biologicalmaterial.

In particular embodiments, the fluid is blood. Methods of the inventionallow for bacteria in a blood sample to be isolated and detected at alevel as low as or even lower than 1 CFU/ml. Blood may be collected in acontainer, such as a blood collection tube (e.g., VACUTAINER, test tubespecifically designed for venipuncture, commercially available fromBecton, Dickinson and company). In certain embodiments, a solution isadded that prevents or reduces aggregation of endogenous aggregatingfactors, such as heparin in the case of blood.

The blood sample is then mixed with compositions as described above togenerate a mixture that is allowed to incubate such that thecompositions bind to at least one bacterium in the blood sample. Thetype or types of bacteria that will bind compositions of the inventionwill depend on the design of the composition, i.e., which antibodyconjugated particles are used. The mixture is allowed to incubate for asufficient time to allow for the composition to bind to the pathogen inthe blood. The process of binding the composition to the pathogenassociates a magnetic moment with the pathogen, and thus allows thepathogen to be manipulated through forces generated by magnetic fieldsupon the attached magnetic moment.

In general, incubation time will depend on the desired degree of bindingbetween the pathogen and the compositions of the invention (e.g., theamount of moment that would be desirably attached to the pathogen), theamount of moment per target, the amount of time of mixing, the type ofmixing, the reagents present to promote the binding and the bindingchemistry system that is being employed. Incubation time can be anywherefrom about 5 seconds to a few days. Exemplary incubation times rangefrom about 10 seconds to about 2 hours. Binding occurs over a wide rangeof temperatures, generally between 15° C. and 40° C.

In certain embodiments, a buffer solution is added to the sample alongwith the compositions of the invention. An exemplary buffer includesTris(hydroximethyl)-aminomethane hydrochloride at a concentration ofabout 75 mM. It has been found that the buffer composition, mixingparameters (speed, type of mixing, such as rotation, shaking etc., andtemperature) influence binding. It is important to maintain osmolalityof the final solution (e.g., blood+buffer) to maintain high labelefficiency. In certain embodiments, buffers used in methods of theinvention are designed to prevent lysis of blood cells, facilitateefficient binding of targets with magnetic particles and to reduceformation of particle aggregates. It has been found that the buffersolution containing 300 mM NaCl, 75 mM Tris-HCl pH 8.0 and 0.1% Tween 20meets these design goals.

Without being limited by any particular theory or mechanism of action,it is believed that sodium chloride is mainly responsible formaintaining osmolality of the solution and for the reduction ofnon-specific binding of magnetic particle through ionic interaction.

Tris(hydroximethyl)-aminomethane hydrochloride is a well establishedbuffer compound frequently used in biology to maintain pH of a solution.It has been found that 75 mM concentration is beneficial and sufficientfor high binding efficiency. Likewise, Tween 20 is widely used as a milddetergent to decrease nonspecific attachment due to hydrophobicinteractions. Various assays use Tween 20 at concentrations ranging from0.01% to 1%. The 0.1% concentration appears to be optimal for theefficient labeling of bacteria, while maintaining blood cells intact.

Additional compounds can be used to modulate the capture efficiency byblocking or reducing non-specific interaction with blood components andeither magnetic particles or pathogens. For example, chelatingcompounds, such as EDTA or EGTA, can be used to prevent or minimizeinteractions that are sensitive to the presence of Ca²⁺ or Mg²⁺ ions.

An alternative approach to achieve high binding efficiency whilereducing time required for the binding step is to use static mixer, orother mixing devices that provide efficient mixing of viscous samples athigh flow rates, such as at or around 5 mL/min. In one embodiment, thesample is mixed with binding buffer in ratio of, or about, 1:1, using amixing interface connector. The diluted sample then flows through amixing interface connector where it is mixed with target-specificnanoparticles. Additional mixing interface connectors providing mixingof sample and antigen-specific nanoparticles can be attached downstreamto improve binding efficiency. The combined flow rate of the labeledsample is selected such that it is compatible with downstreamprocessing.

After binding of the compositions to the pathogen in the sample to formpathogen/magnetic particle complexes, a magnetic field is applied to themixture to capture the complexes on a surface. Components of the mixturethat are not bound to magnetic particles will not be affected by themagnetic field and will remain free in the mixture. Methods andapparatuses for separating target/magnetic particle complexes from othercomponents of a mixture are known in the art. For example, a steel meshmay be coupled to a magnet, a linear channel or channels may beconfigured with adjacent magnets, or quadrapole magnets with annularflow may be used. Other methods and apparatuses for separatingtarget/magnetic particle complexes from other components of a mixtureare shown in Rao et al. (U.S. Pat. No. 6,551,843), Liberti et al. (U.S.Pat. No. 5,622,831), Hatch et al. (U.S. Pat. No. 6,514,415), Benjamin etal. (U.S. Pat. No. 5,695,946), Liberti et al. (U.S. Pat. No. 5,186,827),Wang et al. (U.S. Pat. No. 5,541,072), Liberti et al. (U.S. Pat. No.5,466,574), and Terstappen et al. (U.S. Pat. No. 6,623,983), the contentof each of which is incorporated by reference herein in its entirety.

In certain embodiments, the magnetic capture is achieved at highefficiency by utilizing a flow-through capture cell with a number ofstrong rare earth bar magnets placed perpendicular to the flow of thesample. When using a flow chamber with flow path cross-section 0.5 mm×20mm (h×w) and 7 bar NdFeB magnets, the flow rate could be as high as 5mL/min or more, while achieving capture efficiency close to 100%.

The above described type of magnetic separation produces efficientcapture of a target analyte and the removal of a majority of theremaining components of a sample mixture. However, such a processproduces a sample that contains a very high percent of magneticparticles that are not bound to target analytes because the magneticparticles are typically added in excess, as well as non-specific targetentities. Non-specific target entities may for example be bound at amuch lower efficiency, for example 1% of the surface area, while atarget of interest might be loaded at 50% or nearly 100% of theavailable surface area or available antigenic cites. However, even 1%loading may be sufficient to impart force necessary for trapping in amagnetic gradient flow cell or sample chamber.

For example, in the case of immunomagnetic binding of bacteria or fungiin a blood sample, the sample may include: bound targets at aconcentration of about 1/mL or a concentration less than about 10⁶/mL;background particles at a concentration of about 10⁷/ml to about10¹⁰/ml; and non-specific targets at a concentration of about 10/ml toabout 10⁵/ml.

The presence of magnetic particles that are not bound to target analytesand non-specific target entities on the surface that includes thetarget/magnetic particle complexes interferes with the ability tosuccessfully detect the target of interest. The magnetic capture of theresulting mix, and close contact of magnetic particles with each otherand bound targets, result in the formation of aggregate that is hard todispense and which might be resistant or inadequate for subsequentprocessing or analysis steps. In order to remove magnetic particles thatare not bound to target analytes and non-specific target entities, thesurface may be washed with a wash solution that reduces particleaggregation, thereby isolating target/magnetic particle complexes fromthe magnetic particles that are not bound to target analytes andnon-specific target entities. The wash solution minimizes the formationof the aggregates.

Any wash solution that imparts a net negative charge to the magneticparticle that is not sufficient to disrupt interaction between thetarget-specific moiety of the magnetic particle and the target analytemay be used. Without being limited by any particular theory or mechanismof action, it is believed that attachment of the negatively chargedmolecules in the wash solution to magnetic particles provides netnegative charge to the particles and facilitates dispersal ofnon-specifically aggregated particles. At the same time, the netnegative charge is not sufficient to disrupt strong interaction betweenthe target-specific moiety of the magnetic particle and the targetanalyte (e.g., an antibody-antigen interaction). Exemplary solutionsinclude heparin, Tris-HCl, Tris-borate-EDTA (TBE), Tris-acetate-EDTA(TAE), Tris-cacodylate, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid), PBS (phosphatebuffered saline), PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid), MES(2-N-morpholino)ethanesulfonic acid), Tricine(N-(Tri(hydroximethyl)methyl)glycine), and similar buffering agents. Incertain embodiments, only a single wash cycle is performed. In otherembodiments, more than one wash cycle is performed.

In particular embodiments, the wash solution includes heparin. Forembodiments in which the body fluid sample is blood, the heparin alsoreduces probability of clotting of blood components after magneticcapture. The bound targets are washed with heparin-containing buffer 1-3times to remove blood components and to reduce formation of aggregates.

In addition to the above described methods, compositions of theinvention may be applied or adapted to other methods for isolatingpathogens using other methods, including the methods described inco-owned U.S. publication nos. 2011/0263833, 2011/0262925, 2011/0262932,2011/0262933, 2011/0262926, and 2011/0262927, the entireties of whichare incorporated by reference.

Once the target/magnetic particle complexes are isolated, the target maybe analyzed by a multitude of existing technologies, such as miniatureNMR, Polymerase Chain Reaction (e.g., PCR, digital PCR, real-time orquantitative PCR), Enzyme Immunoassay (EIA), mass spectrometry,fluorescent labeling and visualization using microscopic observation,fluorescent in situ hybridization (FISH), growth-based antibioticsensitivity tests, and variety of other methods that may be conductedwith purified target without significant contamination from other samplecomponents. In one embodiment, isolated bacteria are eluted from themagnetic particles and are lysed with a chaotropic solution, and DNA isbound to DNA extraction resin. After washing of the resin, the bacterialDNA is eluted and used in quantitative RT-PCR to detect the presence ofa specific species, and/or, subclasses of bacteria.

In another embodiment, captured bacteria is removed from the magneticparticles to which they are bound and the processed sample is mixed withfluorescent labeled antibodies specific to the bacteria or fluorescentGram stain. After incubation, the reaction mixture is filtered through0.2 μm to 1.0 μm filter to capture labeled bacteria while allowingmajority of free particles and fluorescent labels to pass through thefilter. Bacteria is visualized on the filter using microscopictechniques, e.g. direct microscopic observation, laser scanning or otherautomated methods of image capture. The presence of bacteria is detectedthrough image analysis. After the positive detection by visualtechniques, the bacteria can be further characterized using PCR orgenomic methods.

Detection of pathogens of interest can be performed by use of nucleicacid probes following procedures which are known in the art. Suitableprocedures for detection of pathogens using nucleic acid probes aredescribed, for example, in Stackebrandt et al. (U.S. Pat. No.5,089,386), King et al. (WO 90/08841), Foster et al. (WO 92/15883), andCossart et al. (WO 89/06699), each of which is hereby incorporated byreference.

A suitable nucleic acid probe assay generally includes sample treatmentand lysis, hybridization with selected probe(s), hybrid capture, anddetection. Lysis of the pathogens (e.g. bacteria or fungi) is necessaryto release the nucleic acid for the probes. The nucleic acid targetmolecules are released by treatment with any of a number of lysisagents, including alkali (such as NaOH), guanidine salts (such asguanidine thiocyanate), enzymes (such as lysozyme, mutanolysin andproteinase K), and detergents. Lysis of the pathogen, therefore,releases both DNA and RNA, particularly ribosomal RNA and chromosomalDNA both of which can be utilized as the target molecules withappropriate selection of a suitable probe. Use of rRNA as the targetmolecule(s), may be advantageous because rRNAs constitute a significantcomponent of cellular mass, thereby providing an abundance of targetmolecules. The use of rRNA probes also enhances specificity for thebacteria of interest, that is, positive detection without undesirablecross-reactivity which can lead to false positives or false detection.

Hybridization includes addition of the specific nucleic acid probes. Ingeneral, hybridization is the procedure by which two partially orcompletely complementary nucleic acids are combined, under definedreaction conditions, in an anti-parallel fashion to form specific andstable hydrogen bonds. The selection or stringency of thehybridization/reaction conditions is defined by the length and basecomposition of the probe/target duplex, as well as by the level andgeometry of mis-pairing between the two nucleic acid strands. Stringencyis also governed by such reaction parameters as temperature, types andconcentrations of denaturing agents present and the type andconcentration of ionic species present in the hybridization solution.

The hybridization phase of the nucleic acid probe assay is performedwith a single selected probe or with a combination of two, three or moreprobes. Probes are selected having sequences which are homologous tounique nucleic acid sequences of the target organism. In general, afirst capture probe is utilized to capture formed hybrid molecules. Thehybrid molecule is then detected by use of antibody reaction or by useof a second detector probe which may be labelled with a radioisotope(such as phosphorus-32) or a fluorescent label (such as fluorescein) orchemiluminescent label.

Detection of pathogen of interest can also be performed by use of PCRtechniques. A suitable PCR technique is described, for example, inVerhoef et al. (WO 92/08805). Such protocols may be applied directly tothe pathogen captured on the magnetic particles. The pathogen iscombined with a lysis buffer and collected nucleic acid target moleculesare then utilized as the template for the PCR reaction.

In certain embodiments, nucleic acids derived from the captured pathogenare analyzed in a multiplex reaction in order to rapidly detect two ormore pathogens present in the sample. Any multiplex reaction known inthe art may be used, such as multiplex ELISA, multiplex sequencing,multiplex probe hybridization, etc. In certain embodiments, multiplexreactions may involve the amplification and quantification of two ormore targets in the same reaction volume. Typical multiplex reactionsinvolve PCR, qPCR (real-time PCR), and sequencing.

Multiplex PCR refers to the use of more than one primer pair in a singlereaction vessel in order to amplify more than one target sequence. Inthe case of real-time PCR, more than one primer pair/probe set isutilized. Multiplex PCR allows for simultaneous detection of differenttargets in the same reaction. The target sequences may be identified byan identifiable label (e.g. fluorescent probe) or by subsequentsequencing. Multiplex real-time PCR uses multiple probe-based assays, inwhich each assay has a specific probe labeled with a unique fluorescentdye, resulting in different observed colors for each assay. Real-timePCR instruments can discriminate between the fluorescence generated fromdifferent dyes. Different probes are labeled with different dyes thateach have unique emission spectra. Spectral signals are collected withdiscrete optics, passed through a series of filter sets, and collectedby an array of detectors. Spectral overlap between dyes is corrected byusing pure dye spectra to deconvolute the experimental data by matrixalgebra. An overview of real-time PCR techniques is described in detailin Elnifro, Elfath M., et al. “Multiplex PCR: optimization andapplication in diagnostic virology.” Clinical Microbiology Reviews 13.4(2000): 559-570. Multiplex PCR reaction techniques are described in U.S.Publication Nos. 20130059762, and 2005/0026144 as well as Carroll, N.M., E. E. Jaeger, et al. (2000). “Detection of and discriminationbetween gram-positive and gram-negative bacteria in intraocular samplesby using nested PCR.” J Clin Microbiol 38(5): 1753-1757, and Klaschik,S., L. E. Lehmann, et al. (2002). “Real-time PCR for detection anddifferentiation of gram-positive and gram-negative bacteria.” J ClinMicrobiol 40(11): 4304-4307.

Multiplex sequencing involves the simultaneous sequencing of multipletarget sequences in a single sequencing run. Multiplex sequencing allowsfor differentiation between target sequences of different pathogens in asample and differentiation between nucleic acid sequences of pooledsamples (e.g. differentiate between two or more patient samples). Inorder to differentiate between target sequences, one or more differentbarcodes may be introduced to the nucleic acid of a sample. Earlymultiplex sequencing is described in more detail in G. M. Church in U.S.Pat. No. 4,942,124 and further by G. M. Church and S. Kieffer-Higgins inU.S. Pat. No. 5,149,625. Multiplex sequencing of multiple samplesinvolves sequencing of a plurality of template nucleic acid moleculesfrom different samples at the same time on the same platform byattaching a unique oligonucleotide sequence (i.e., a bar code) to thetemplate nucleic acid molecules from different samples prior to poolingand sequencing of the template molecules. The bar code allows fortemplate nucleic acid sequences from different samples to bedifferentiated from each other. Once bar coded, template molecules fromdifferent samples may be pooled and sequenced at the same time on thesame platform. Because the bar code on each template molecule issequenced as part of the sequencing reaction, the bar code is acomponent of the sequence data, and thus the sequence data for differentsamples is always associated with the sample from which it originated.Due to the association in the sequence data, the sequence data from thepooled samples may be separated after sequencing has occurred andcorrelated back to the sample from which it originated.

Any sequencing technique (for singleplex and multiplex assays) may beutilized to identify isolated pathogens by their nucleic acid extracts.Suitable sequencing techniques include, for example, classic dideoxysequencing reactions (Sanger method) using labeled terminators orprimers and gel separation in slab or capillary, sequencing by synthesisusing reversibly terminated labeled nucleotides, pyrosequencing, 454sequencing, allele specific hybridization to a library of labeledoligonucleotide probes, sequencing by synthesis using allele specifichybridization to a library of labeled clones that is followed byligation, real time monitoring of the incorporation of labelednucleotides during a polymerization step, polony sequencing, and SOLiDsequencing. Sequencing of separated molecules has more recently beendemonstrated by sequential or single extension reactions usingpolymerases or ligases as well as by single or sequential differentialhybridizations with libraries of probes.

For detection of the selected pathogens by use of antibodies, isolatedpathogen are contacted with antibodies specific to the pathogen ofinterest. As noted above, either polyclonal or monoclonal antibodies canbe utilized, but in either case have affinity for the particularbacteria to be detected. These antibodies, will adhere/bind to materialfrom the specific target bacteria. With respect to labeling of theantibodies, these are labeled either directly or indirectly with labelsused in other known immunoassays. Direct labels may include fluorescent,chemiluminescent, bioluminescent, radioactive, metallic, biotin orenzymatic molecules. Methods of combining these labels to antibodies orother macromolecules are well known to those in the art. Examplesinclude the methods of Hijmans, W. et al. (1969), Clin. Exp. Immunol. 4,457-, for fluorescein isothiocyanate, the method of Goding, J. W.(1976), J. Immunol. Meth. 13, 215-, for tetramethylrhodamineisothiocyanate, and the method of Ingrall, E. (1980), Meth. in Enzymol.70, 419-439 for enzymes.

These detector antibodies may also be labeled indirectly. In this casethe actual detection molecule is attached to a secondary antibody orother molecule with binding affinity for the anti-bacteria cell surfaceantibody. If a secondary antibody is used it is preferably a generalantibody to a class of antibody (IgG and IgM) from the animal speciesused to raise the anti-bacteria cell surface antibodies. For example,the second antibody may be conjugated to an enzyme, either alkalinephosphatase or to peroxidase. To detect the label, after the bacteria ofinterest is contacted with the second antibody and washed, the isolatedcomponent of the sample is immersed in a solution containing achromogenic substrate for either alkaline phosphatase or peroxidase. Achromogenic substrate is a compound that can be cleaved by an enzyme toresult in the production of some type of detectable signal which onlyappears when the substrate is cleaved from the base molecule. Thechromogenic substrate is colorless, until it reacts with the enzyme, atwhich time an intensely colored product is made. Thus, material from thebacteria colonies adhered to the membrane sheet will become an intenseblue/purple/black color, or brown/red while material from other colonieswill remain colorless. Examples of detection molecules includefluorescent substances, such as 4-methylumbelliferyl phosphate, andchromogenic substances, such as 4-nitrophenylphosphate,3,3′,5,5′-tetramethylbenzidine and2,2′-azino-di-[3-ethelbenz-thiazoliane sulfonate (6)]. In addition toalkaline phosphatase and peroxidase, other useful enzymes includeβ-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase,α-mannosidase, galactose oxidase, glucose oxidase and hexokinase.

Detection of bacteria of interest using NMR may be accomplished asfollows. In the use of NMR as a detection methodology, in which a sampleis delivered to a detector coil centered in a magnet, the target ofinterest, such as a magnetically labeled bacterium, may be delivered bya fluid medium, such as a fluid substantially composed of water. In sucha case, the magnetically labeled target may go from a region of very lowmagnetic field to a region of high magnetic field, for example, a fieldproduced by an about 1 to about 2 Tesla magnet. In this manner, thesample may traverse a magnetic gradient, on the way into the magnet andon the way out of the magnet. As may be seen via equations 1 and 2below, the target may experience a force pulling into the magnet in thedirection of sample flow on the way into the magnet, and a force intothe magnet in the opposite direction of flow on the way out of themagnet. The target may experience a retaining force trapping the targetin the magnet if flow is not sufficient to overcome the gradient force.

m dot(del B)=F  Equation 1

v _(t) =−F/(6*p*n*r)  Equation 2

where n is the viscosity, r is the particle diameter, F is the vectorforce, B is the vector field, and m is the vector moment of the particle

Magnetic fields on a path into a magnet may be non-uniform in thetransverse direction with respect to the flow into the magnet. As such,there may be a transverse force that pulls targets to the side of acontainer or a conduit that provides the sample flow into the magnet.Generally, the time it takes a target to reach the wall of a conduit isassociated with the terminal velocity and is lower with increasingviscosity. The terminal velocity is associated with the drag force,which may be indicative of creep flow in certain cases. In general, itmay be advantageous to have a high viscosity to provide a higher dragforce such that a target will tend to be carried with the fluid flowthrough the magnet without being trapped in the magnet or against theconduit walls.

Newtonian fluids have a flow characteristic in a conduit, such as around pipe, for example, that is parabolic, such that the flow velocityis zero at the wall, and maximal at the center, and having a paraboliccharacteristic with radius. The velocity decreases in a direction towardthe walls, and it is easier to magnetically trap targets near the walls,either with transverse gradients force on the target toward the conduitwall, or in longitudinal gradients sufficient to prevent target flow inthe pipe at any position. In order to provide favorable fluid drag forceto keep the samples from being trapped in the conduit, it may beadvantageous to have a plug flow condition, wherein the fluid velocityis substantially uniform as a function of radial position in theconduit.

When NMR detection is employed in connection with a flowing sample, thedetection may be based on a perturbation of the NMR water signal causedby a magnetically labeled target (Sillerud et al., JMR (Journal ofMagnetic Resonance), vol. 181, 2006). In such a case, the sample may beexcited at time 0, and after some delay, such as about 50 ms or about100 ms, an acceptable measurement (based on a detected NMR signal) maybe produced. Alternatively, such a measurement may be producedimmediately after excitation, with the detection continuing for someduration, such as about 50 ms or about 100 ms. It may be advantageous todetect the NMR signal for substantially longer time durations after theexcitation.

By way of example, the detection of the NMR signal may continue for aperiod of about 2 seconds in order to record spectral information athigh-resolution. In the case of parabolic or Newtonian flow, theperturbation excited at time 0 is typically smeared because the wateraround the perturbation source travels at different velocity, dependingon radial position in the conduit. In addition, spectral information maybe lost due to the smearing or mixing effects of the differential motionof the sample fluid during signal detection. When carrying out an NMRdetection application involving a flowing fluid sample, it may beadvantageous to provide plug-like sample flow to facilitate desirableNMR contrast and/or desirable NMR signal detection.

Differential motion within a flowing Newtonian fluid may havedeleterious effects in certain situations, such as a situation in whichspatially localized NMR detection is desired, as in magnetic resonanceimaging. In one example, a magnetic object, such as a magneticallylabeled bacterium, is flowed through the NMR detector and its presenceand location are detected using MRI techniques. The detection may bepossible due to the magnetic field of the magnetic object, since thisfield perturbs the magnetic field of the fluid in the vicinity of themagnetic object. The detection of the magnetic object is improved if thefluid near the object remains near the object. Under these conditions,the magnetic perturbation may be allowed to act longer on any givenvolume element of the fluid, and the volume elements of the fluid soaffected will remain in close spatial proximity. Such a stronger, morelocalized magnetic perturbation will be more readily detected using NMRor MRI techniques.

If a Newtonian fluid is used to carry the magnetic objects through thedetector, the velocity of the fluid volume elements will depend onradial position in the fluid conduit. In such a case, the fluid near amagnetic object will not remain near the magnetic object as the objectflows through the detector. The effect of the magnetic perturbation ofthe object on the surrounding fluid may be smeared out in space, and thestrength of the perturbation on any one fluid volume element may bereduced because that element does not stay within range of theperturbation. The weaker, less-well-localized perturbation in the samplefluid may be undetectable using NMR or MRI techniques.

Certain liquids, or mixtures of liquids, exhibit non-parabolic flowprofiles in circular conduits. Such fluids may exhibit non-Newtonianflow profiles in other conduit shapes. The use of such a fluid may proveadvantageous as the detection fluid in an application employing anNMR-based detection device. Any such advantageous effect may beattributable to high viscosity of the fluid, a plug-like flow profileassociated with the fluid, and/or other characteristic(s) attributed tothe fluid that facilitate detection. As an example, a shear-thinningfluid of high viscosity may exhibit a flow velocity profile that issubstantially uniform across the central regions of the conduitcross-section. The velocity profile of such a fluid may transition to azero or very low value near or at the walls of the conduit, and thistransition region may be confined to a very thin layer near the wall.

Not all fluids, or all fluid mixtures, are compatible with the NMRdetection methodology. In one example, a mixture of glycerol and watercan provide high viscosity, but the NMR measurement is degraded becauseseparate NMR signals are detected from the water and glycerol moleculesmaking up the mixture. This can undermine the sensitivity of the NMRdetector. In another example, the non-water component of the fluidmixture can be chosen to have no NMR signal, which may be achieved byusing a perdeuterated fluid component, for example, or using aperfluorinated fluid component. This approach may suffer from the lossof signal intensity since a portion of the fluid in the detection coildoes not produce a signal.

Another approach may be to use a secondary fluid component thatconstitutes only a small fraction of the total fluid mixture. Such alow-concentration secondary fluid component can produce an NMR signalthat is of negligible intensity when compared to the signal from themain component of the fluid, which may be water. It may be advantageousto use a low-concentration secondary fluid component that does notproduce an NMR signal in the detector. For example, a perfluorinated orperdeuterated secondary fluid component may be used. The fluid mixtureused in the NMR detector may include one, two, or more than twosecondary components in addition to the main fluid component. The fluidcomponents employed may act in concert to produce the desired fluid flowcharacteristics, such as high-viscosity and/or plug flow. The fluidcomponents may be useful for providing fluid characteristics that areadvantageous for the performance of the NMR detector, for example byproviding NMR relaxation times that allow faster operation or highersignal intensities.

A non-Newtonian fluid may provide additional advantages for thedetection of objects by NMR or MRI techniques. As one example, theobjects being detected may all have substantially the same velocity asthey go through the detection coil. This characteristic velocity mayallow simpler or more robust algorithms for the analysis of thedetection data. As another example, the objects being detected may havefixed, known, and uniform velocity. This may prove advantageous indevices where the position of the detected object at later times isneeded, such as in a device that has a sequestration chamber orsecondary detection chamber down-stream from the NMR or MRI detectioncoil, for example.

In an exemplary embodiment, sample delivery into and out of a 1.7 Tcylindrical magnet using a fluid delivery medium containing 0.1% to 0.5%Xanthan gum in water was successfully achieved. Such delivery issuitable to provide substantially plug-like flow, high viscosity, suchas from about 10 cP to about 3000 cP, and good NMR contrast in relationto water. Xanthan gum acts as a non-Newtonian fluid, havingcharacteristics of a non-Newtonian fluid that are well known in the art,and does not compromise NMR signal characteristics desirable for gooddetection in a desirable mode of operation.

In certain embodiments, methods of the invention are useful for directdetection of bacteria from blood. Such a process is described here.Sample is collected in sodium heparin tube by venipuncture, acceptablesample volume is about 1 mL to 10 mL. Sample is diluted with bindingbuffer and superparamagnetic particles having target-specific bindingmoieties are added to the sample, followed by incubation on a shakingincubator at 37° C. for about 30 min to 120 min. Alternative mixingmethods can also be used. In a particular embodiment, sample is pumpedthrough a static mixer, such that reaction buffer and magnetic particlesare added to the sample as the sample is pumped through the mixer. Thisprocess allows for efficient integration of all components into a singlefluidic part, avoids moving parts and separate incubation vessels andreduces incubation time.

Capture of the labeled targets allows for the removal of bloodcomponents and reduction of sample volume from 30 mL to 5 mL. Thecapture is performed in a variety of magnet/flow configurations. Incertain embodiments, methods include capture in a sample tube on ashaking platform or capture in a flow-through device at flow rate of 5mL/min, resulting in total capture time of 6 min.

After capture, the sample is washed with wash buffer including heparinto remove blood components and free particles. The composition of thewash buffer is optimized to reduce aggregation of free particles, whilemaintaining the integrity of the particle/target complexes.

The detection method is based on a miniature NMR detector tuned to themagnetic resonance of water. When the sample is magnetically homogenous(no bound targets), the NMR signal from water is clearly detectable andstrong. The presence of magnetic material in the detector coil disturbsthe magnetic field, resulting in reduction in water signal. One of theprimary benefits of this detection method is that there is no magneticbackground in biological samples which significantly reduces therequirements for stringency of sample processing. In addition, since thedetected signal is generated by water, there is a built-in signalamplification which allows for the detection of a single labeledbacterium.

The above described methods for isolating a pathogen may be carried outin a self-contained macrofluidic and/or microfluidic system.Microfluidic substrates or cartridges are known in the art and includeone or more chambers, channels, reservoirs, mixers, and traps configuredto carry out reactions within the substrate or cartridge. A benefit ofmicrofluidic substrates is that there is no need for external tubing tointroduce the various products required for the reaction. Channels ofmicrofluidic system connect and interconnect various components, suchas, through holes, slides, foil caps, alignment features, liquid andlyophilized reagent storage chambers, reagent release chambers, pumps,metering chambers, lyophilized cake reconstitution chambers, ultrasonicchambers, joining and mixing chambers, mixing elements such as a mixingpaddle and other mixing gear, membrane regions, filtration regions,venting elements, heating elements, magnetic traps/chambers, reactionchambers, waste chambers, membrane regions, thermal transfer regions,anodes, cathodes, and detection regions, drives, plugs, piercing blades,valve lines, valve structures, assembly features such as o-rings,instrument interface regions, cartridge/vessel interfaces, one or moreneedles associated with the sample interface, optical windows, thermalwindows, and detection regions. Pressure sources can be utilized todrive flow and introduce fluid into and out of the microfluidic system.Microfluidic cartridges and microfluidic components can be designedusing any technique known in the art, for example the microfluidicfabrication techniques in Lab on a Chip Technology: Fabrication andMicrofluidics, Vol. 1, Herold & Rasooley, Caister Academic Press (2009).An exemplary macrofluidic system and microfluidic system that can beused to carry out methods of the invention is reported in co-owned andco-assigned U.S. Provisional App. No. 61/739,644, filed Dec. 19, 2012,the entirety of which is incorporated by reference.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES Example 1 Sample

Blood samples from healthy volunteers were spiked with clinicallyrelevant concentrations of bacteria (1-10 CFU/mL) including bothlaboratory strains and clinical isolates of the bacterial species mostfrequently found in bloodstream infections.

Example 2 Antibody Preparation

In order to generate polyclonal, pan-Gram-positive bacteria-specificIgG, a goat was immunized by first administering bacterial antigenssuspended in complete Freund's adjuvant intra lymph node, followed bysubcutaneous injection of bacterial antigens in incomplete Freund'sadjuvant in 2 week intervals. The antigens were prepared for antibodyproduction by growing bacteria to exponential phase (OD₆₀₀=0.4-0.8).Following harvest of the bacteria by centrifugation, the bacteria wasinactivated using formalin fixation in 4% formaldehyde for 4 hr at 37°C. After 3 washes of bacteria with PBS (15 min wash, centrifugation for20 min at 4000 rpm) the antigen concentration was measured using BCAassay and the antigen was used at 1 mg/mL for immunization. In order togenerate Gram-positive bacteria—specific IgG, several bacterial specieswere used for inoculation: Staphylococcus aureus, Staphylococcusepidermidis, Enterococcus faecium and Enterococcus fecalis.

The immune serum was purified using affinity chromatography on a proteinG sepharose column (GE Healthcare), and reactivity was determined usingELISA. Antibodies cross-reacting with Gram-negative bacteria and fungiwere removed by absorption of purified IgG with formalin-fixedGram-negative bacteria and fungi. The formalin-fixed organisms wereprepared similar to as described above and mixed with IgG. Afterincubation for 2 hrs at room temperature, the preparation wascentrifuged to remove bacteria. Final antibody preparation was clarifiedby centrifugation and used for the preparation of antigen-specificmagnetic particles.

Pan-Gram-negative IgG were generated in a similar fashion usinginactivated Enterobacter cloacae, Pseudomonas aeruginosa, Serratiamarcescens and other gram-negative bacteria as immunogens. The IgGfraction of serum was purified using protein-G affinity chromatographyas described above.

Similarly, target specific antibodies were generated by inoculation ofgoats using formalin-fixed bacteria, immunization was performed with 2or more closely related organisms.

Example 3 Preparation of Antigen-Specific Magnetic Particles

Superparamagnetic particles were synthesized by encapsulating iron oxidenanoparticles (5-15 nm diameter) in a latex core and labeling with goatIgG. Ferrofluid containing nanoparticles in organic solvent wasprecipitated with ethanol, nanoparticles were resuspended in aqueoussolution of styrene and surfactant Hitenol BC-10, and emulsified usingsonication. The mixture was allowed to equilibrate overnight withstirring and filtered through 1.2 and 0.45 μm filters to achieve uniformmicelle size. Styrene, acrylic acid and divynilbenzene were added incarbonate buffer at pH 9.6. The polymerization was initiated in amixture at 70° C. with the addition of K₂S₂O₈ and the reaction wasallowed to complete overnight. The synthesized particles were washed 3times with 0.1% SDS using magnetic capture, filtered through 1.2, 0.8,and 0.45 μm filters and used for antibody conjugation.

The production of particles resulted in a distribution of sizes that maybe characterized by an average size and a standard deviation. In thecase of labeling and extracting of bacteria from blood, the average sizefor optimal performance was found to be between 100 and 350 nm, forexample between 200 nm to 250 nm.

The purified IgG were conjugated to prepared particles using standardEDC/sulfo-NHS chemistry. After conjugation, the particles wereresuspended in 0.1% BSA which is used to block non-specific bindingsites on the particle and to increase the stability of particlepreparation.

Example 4 Labeling of Rare Cells Using Excess of Magnetic Nanoparticles

Bacteria, present in blood during blood-stream infection, weremagnetically labeled using the superparamagnetic particles prepared inExample 3 above. The spiked samples as described in Example 1 werediluted 3-fold with a Tris-based binding buffer and target-specificparticles, followed by incubation on a shaking platform at 37° C. for upto 2 hr. The optimal concentration of particles was determined bytitration and was found to be in the range between 1×10⁸ and 5×10¹⁰particle/mL. After incubation, the labeled targets were magneticallyseparated followed by a wash step designed to remove blood products. Seeexample 5 below.

Example 5 Magnetic Capture of Bound Bacteria

Blood including the magnetically labeled target bacteria and excess freeparticles were injected into a flow-through capture cell with a numberof strong rare earth bar magnets placed perpendicular to the flow of thesample. With using a flow chamber with flow path cross-section 0.5 mm×20mm (h×w) and 7 bar NdFeB magnets, a flow rate as high as 5 mL/min wasachieved. After flowing the mixture through the channel in the presenceof the magnet, a wash solution including heparin was flowed through thechannel. The bound targets were washed with heparin-containing bufferone time to remove blood components and to reduce formation of magneticparticle aggregates. In order to effectively wash bound targets, themagnet was removed and captured magnetic material was resuspended inwash buffer, followed by re-application of the magnetic field andcapture of the magnetic material in the same flow-through capture cell.

Removal of the captured labeled targets was possible after movingmagnets away from the capture chamber and eluting with flow of buffersolution.

What is claimed is:
 1. A method for isolating at least one pathogen froma heterogeneous sample, the method comprising: obtaining a heterogeneoussample comprising at least one pathogen; incubating the sample with acomposition comprising a plurality of sets of magnetic particlesspecific to one or more pathogens, wherein members of a first set ofmagnetic particles are conjugated to a first class of capture moiety,and members of a second set of magnetic particles are conjugated to asecond class of capture moiety; separating particle bound pathogen fromother components in the sample.
 2. The method of claim 1, wherein thefirst and second classes of capture moieties are selected from the groupconsisting of antibodies, lectins, bacteriophages, antimicrobial agents,and oligonucleotides.
 3. The method of claim 1, wherein each set ofmagnetic particles is present at a concentration designed for detectionof a specific pathogen in said sample.
 4. The method of claim 3, whereinthe first and second set of magnetic particles are provided at differentconcentrations.
 5. The method of claim 1, wherein the pathogen isselected from the group consisting of fungi, bacteria and a combinationthereof.
 6. The method of claim 5, wherein the fungi comprises two ormore species of fungi selected from the group consisting of the Candidagenus, Aspergillus genus, and Cryptococcus genus.
 7. The method of claim5, wherein the bacteria comprise one or species of bacteria selectedfrom the group consisting of Staphylococcus genus, Enterobacteriaceaegenus, Acinetobacter genus, Strenotrophomonas genus, Pseudomanos genus,Neisseria genus, Clostridium genus, and Enterococcus genus.
 8. Themethod of claim 1, wherein the first and second classes are different.9. The method of claim 1, wherein the first and second classes are thesame.
 10. The method of claim 1, wherein members of the first class aredifferent.
 11. The method of claim 1, wherein members of the first classare the same.
 12. The method of claim 1, wherein members of the secondclass are different.
 13. The method of claim 1, wherein members of thesecond class are the same.
 14. The method of claim 1, further comprisingidentifying the pathogen.
 15. The method of claim 1, further comprisinglysing the separated pathogen to release nucleic acid.
 16. The method ofclaim 14, further comprising conducting an assay on the nucleic acid toidentify the pathogen.
 17. The method of claim 16, wherein the assay isselected from the group consisting of a sequencing reaction and apolymerase chain reaction.
 18. The method of claim 16, wherein the assayis a multiplex assay.